The present and well-known technology for DNA oligonucleotide (oligo) detection is reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The techniques are essential for identifying virus genes in one sample. Firstly, the RT-PCR technique is a genetic diagnostic technique based on cloning expressed genes by reverse transcribing the RNA of virus into its DNA complement and amplification of the complement DNA (c-DNA) via thereto-cycling in a thermos cycler. This technology involves the sophistically-designed primers for efficient amplification via nucleic acid hybridization. The readout is done by using gel electrophoresis. The whole process may require 1-3 days for accurate results. The ELISA technique is a solid-stale colorimetric immunoassay, which is based on antibody-antigen interaction via the viruses surface protein. Initially, the antigen is anchored on the substrate and a specific type of antibody linked with enzyme is added to the substrate. The interaction of the antigen and the antibody will form a complex to produce color change. As a result, the antibody expressed on the surface of virus can be identified by such technique. However, PCR requires well-trained personnel for operating the thermocycler and the amplification process is relatively time-consuming. The amplification steps are prone to contamination during successive steps. On the other hand, ELISA kits are commercialized and available from many suppliers. The kits consist of necessary chemicals and substrate for testes. However, the procedures of ELISA are laborious and the limit of detection is relatively low (nanomolar range). Owing to these shortcomings, the quest for searching sensitive and quick diagnostic assays is still on-going.
In recent years, luminescent assays are drawing attention because of their high sensitivity and the ease of making portable devices for on-site biodetections. Luminescent assays are divided into homogeneous and heterogeneous assays. Homogeneous assays are liquid phase test, and they are usually performed in micro-centrifuge tubes and simple mixing steps are required to observe the results. On the other hand, heterogeneous assays are more sensitive than homogeneous assay because of the higher binding affinity between the probe and analyte. One of the key features is the use of a solid phase substrate for detection. The results in both assays can be interpreted by using a portable light source and simple optical detectors, such as CMOS or CCDs. Therefore, they are much simpler than PCR and ELISA techniques. Nowadays, downconversion (DC) or downshifting (DS) luminescence-based assays are being reported for rapid luminescent detections. However, such luminescence mechanisms require the use of high energy light sources, such as ultraviolet (UV). It is a common knowledge that UV is harmful to DNAs and it will destroy chemical oligo chain backbones. Moreover, UV will induce autofluorescence, which will contribute to false-positive detection signals. As a result, upconversion luminescence (UCL) assays are developed to overcome the above-mentioned drawbacks. UCL is a unique luminescent phenomenon that involves sequential absorption of lower energy photons to emit a higher energy photon. In this regard, the low energy excitation can reduce the photodamage to biological samples to a minimum. Moreover, it is easier to distinguish the luminescent detection signal because of the large anti-stoke shift and the invisible near infrared (NIR) excitation. Despite UCL requires the use of lasers, the availability of cheap and portable diode lasers has overcome the issue.
The upconversion nanoparticles (UCNPs) can be obtained by hydrothermal method. The advantages are simplicity and ease of manipulation because water dispersible UCNPs with amine (NH2) surface is readily obtained via a one-step hydrothermal method. However, it is relatively time consuming, requiring about 24 h of reaction time for completion, and the resultant NH2-UCNPs are not regular in shape.
The UCNPs BaGdF5:Yb/Er has been disclosed for homogeneous detection of Avian Influenza Virus H7 subtype (Small 2014, 10, 2390-2397) and heterogeneous detection of Ebola virus oligonucleotide (ACS Nano 2016, 10, 598-605). The UCNP of BaGdF5:Yb/Er was synthesized by hydrothermal method, and the detection scheme was suitable for single target only. Since the emission intensity of BaGdF5:Yb/Er is weak and the nanoparticle is not dispersing very well in water, it is difficult to control their position during the fabrication of the microarray for simultaneous detection of multi-targets.
In addition, structural engineering of core-shell upconversion nanoparticles (csUCNPs) has emerged as a powerful means to integrate functionalities and regulate the complex interplay of lanthanide interactions. The csUCNPs can be obtained by thermal decomposition method and co-precipitation synthesis.
The core-shell NaGdF4:Yb/Er@NaGdF4:Yb/Nd has been disclosed for in vitro and in vivo imaging (ACS Nano 2013, 7, 7200-7206), prepared by thermal decomposition method. The limitations of thermal decomposition method disclosed in the ACS Nano 2013 paper mainly arise from the synthetic route that involves the use of excessive chemicals, such as oleylamine, steps for formation of lanthanide trifluoroacetates and the need to filter the unwanted insoluble materials, which will contaminate the reaction medium. Moreover, the high reaction temperature at 310° C. for synthesis of the core-shell NaGdF4:Yb/Er@NaGdF4:Yb/Nd is undesirable.
The core multishell structured nanoparticles of NaGdF4:Yb,Er@NaYF4:Yb@NaGdF4:Yb,Nd and NaGdF4:Yb,Er@NaYF4:Yb@NaGdF4:Yb,Nd@NaYF4@-NaGdF4:Yb,Tm@NaYF4 were prepared by co-precipitation method for in vivo imaging (Angew. Chem. Int Ed. 2016, 128, 2510-2515). The oleate core-UCNPs was first prepared and then purified to grow the multishell UCNPs. The resultant multiple shell UCNPs involved NaGdF4:Yb/Er as core and NaGdF4:Yb/Nd as intermediate shell. However, the size of these UCNPs is about 45-85 nm which is too large for fabrication of microarray.
There are a lot of viruses that infect different human organs and cause diseases. Some fatal viral infections have become tremendous public health issues worldwide. Early diagnosis for adequate treatment is therefore essential for fighting viral infections. Microarray technology involving core-shell UCNPs can solve the limitation of the PCR method and can be effectively applied to molecular medicine. Microarray can be employed to detect multiple viruses simultaneously, serving as a clinical tool for characterizing viral co-infections in patients.